Abstract 351: Developing A Human Bone Marrow Organoid Culture System To Study The Diabetic Immune System

Objectives: There is limited understanding on how diabetes negatively impacts wound healing after ischemic tissue damage. Murine models for limb ischemia and myocardial infarction have demonstrated that recruitment of bone marrow hematopoietic stem cell (HSC)-derived immune cells at all stages of ischemic tissue remodeling and healing. By leveraging our lab’s three-dimensional (3D) bone marrow organoid culture system used to study human HSC, we aim to study the effect of diabetes on immune cell development. Methods: Surgically discarded bone marrow tissue samples from patients undergoing lower extremity amputation for non-healing ulcer or tissue loss was used to develop our bone marrow 3D organoid culture system. Human HSCs are identified using fluorescence-tagged antibodies against cell surface markers. Hematopoietic potential of bone marrow-derived HSCs was tested using colony forming unit assay. In vitro immune cell differentiation into monocyte and macrophage was performed. Bulk RNA libraries were sequenced on the NextSeq 500 system (Illumina) and analysis done on DeBrowser. Results: Our novel human bone marrow co-culture system provides a method for bone marrow cell expansion from primary human bone marrow tissue explants. De novo sprouting cells can be observed growing from bone marrow tissue explants harvested from non-diabetic and diabetic donors. Using fluorescence-activated cell sorting analysis, we identified a trend towards reduced HSC numbers in diabetic when compared to non-diabetic donors. HSC derived from both diabetic and non-diabetic bone marrow tissue explants were able to undergo hematopoiesis and immune cell differentiation. Bulk RNA sequencing demonstrates distinct gene expression profiles of the cells expanded from non-diabetic and diabetic bone marrow cells. Conclusions: Our 3D bone marrow organoid culture system will allow for future comparative studies between the non-diabetic and diabetic immune system. The preliminary data suggest that there are gene expression profile differences between bone marrow progenitor cells from diabetic and non-diabetic donors. These gene expression differences may attribute to alterations in the HSC number and immune cell development observed in diabetes.